PROJECT SUMMARY Ebola viruses and other members of the filovirus family cause severe and often lethal infections. While some progress has been made in regards to the development of experimental countermeasures and insights into the highly pathogenic nature of these viruses, there are still many more unanswered questions, and further advancement is needed towards the development of pan-filovirus therapeutic agents and vaccines. A significant hurdle to research on filoviruses is the accessibility and cost associated with high-containment, biosafety level- 4 laboratories. To partially alleviate this issue, we previously established a replication-defective Ebola virus based on the Zaire ebolavirus (EBOV) genome. This virus, which lacks the essential viral gene VP30 (termed EBOV?VP30), is biologically inert and safe to use outside of highly specialized BSL-4 containment. In engineered cell lines that stably express EBOV VP30, the virus becomes replication-competent; thus it is a perfect EBOV surrogate for in vitro research given that EBOV?VP30 resembles authentic virus in its life cycle, morphology, protein composition, and growth kinetics. After extensive safety testing both in cell culture and in animal models, the CDC and the Office of Science Policy at the NIH classified EBOV?VP30 as a BSL-2 agent and removed the virus from Select Agent regulations. Since then, this in vitro system to study EBOV has been requested by and distributed to several other research laboratories. The next step to advance the EBOV?VP30 system is to develop an EBOV VP30 transgenic animal model to support virus replication. Previously, we generated a transgenic mouse line that expressed EBOV VP30 under the control of the chicken beta-actin promoter (CAG). Although we were able to detect VP30 mRNA in key organs, such as the liver, and functional VP30 protein in cells, such as fibroblasts, we were unable to detect VP30 mRNA and functional protein in monocyte-derived macrophages, the first target of EBOV infection and a cell type essential for virus dissemination throughout the body. Here, we propose to generate a new transgenic mouse line with EBOV VP30 expression under the control of the CD45 promoter, a promoter specific for expression in hematopoietic cells, including monocytes, macrophages, and dendritic cells. Once we confirm the expression and function of VP30 in these cell types, particularly macrophages, we will cross our two different VP30 transgenic lines (CAG and CD45) to generate a double knock-in transgenic mouse line. Once established, we will infect these transgenic mice with mouse-adapted EBOV?VP30 and characterize the phenotype. We will also generate chimeric versions of EBOV?VP30 with glycoproteins from other filoviruses and examine the phenotype of these chimeric viruses in the transgenic mice. After safety testing, this new small animal model will be an ideal in vivo surrogate for the authentic mouse model for EBOV infection. For the first time, a transgenic mouse model will be available that can be used efficiently and safely outside of BSL-4 containment to examine EBOV pathogenesis and accelerate the development and evaluation of countermeasures.